Ensemble and Single-Molecule Analysis of Non-Homologous End Joining in Frog Egg Extracts

Content Provider	Scilit
Author	Graham, Thomas G. W. Walter, Johannes C. Loparo, Joseph J.
Copyright Year	2017
Description	Non-homologous end joining (NHEJ) repairs the majority of DNA double-strand breaks in human cells, yet the detailed order of events in this process has remained obscure. Here, we describe how to employ Xenopus laevis egg extract for the study of NHEJ. The egg extract is easy to prepare in large quantities, and it performs efficient end joining that requires the core end joining proteins Ku, DNA-PKcs, XLF, XRCC4, and DNA ligase IV. These factors, along with the rest of the soluble proteome, are present at endogenous concentrations, allowing mechanistic analysis in a system that begins to approximate the complexity of cellular end joining. We describe an ensemble assay that monitors covalent joining of DNA ends and fluorescence assays that detect joining of single pairs of DNA ends. The latter assay discerns at least two discrete intermediates in the bridging of DNA ends. Book Name: Protein \3- DNA Interactions
Related Links	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404771/pdf
Ending Page	270
Page Count	38
Starting Page	233
DOI	10.1016/bs.mie.2017.03.020
Volume Number	591
Language	English
Publisher	Elsevier BV
Publisher Date	2017-05-15
Access Restriction	Open
Subject Keyword	Book Name: Protein \3- DNA Interactions Cell Biology Nonhomologous End Joining Xenopus Egg Extract Dna Double-strand Break Repair Single-molecule Fluorescence Förster Resonance Energy Transfer
Content Type	Text
Resource Type	Article